MHC-restricted cytotoxic T-lymphocyte assay: an improved method based on normal and SV40-immortalized rabbit epidermal target cells.
Although several techniques are available to evaluate cell-mediated immunity, numerous difficulties have prevented their use in rabbits. Cytotoxic T-lymphocyte (CTL) assays have been used to determine the ex vivo cytolytic activity of CD8+ T-lymphocytes in immunization protocols. However, this assay cannot be performed with rabbit peripheral blood mononuclear cell (PBMC) targets because of their high spontaneous (51)Cr release. To overcome this intrinsic difficulty shown by rabbit cells, syngeneic normal and SV40-immortalized cells were prepared from skin biopsies. The results show that: (i) skin-derived rabbit fibroblasts can be used as target cells after infection with a fowlpox virus recombinant; (ii) SV40-immortalized skin fibroblasts appear to be more appropriate for repeated assays; (iii) antigen-expanded T-cells and fresh PBMCs can be used as effectors with a similar efficiency; and (iv) dissociation of adherent skin fibroblast target cells with EDTA is to be preferred over TrypLE enzymatic treatment.
Pozzi
Eleana
E
Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy.
Zanotto
Carlo
C
Pacchioni
Sole
S
De Giuli Morghen
Carlo
C
Radaelli
Antonia
A
D000818:Animals; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003601:Cytotoxicity Tests, Immunologic; D000078404:Epidermal Cells; D004817:Epidermis / Q000276:immunology* / Q000821:virology*; D005347:Fibroblasts / Q000276:immunology / Q000821:virology; D005587:Fowlpox virus / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation; D008285:Major Histocompatibility Complex / Q000502:physiology*; D011817:Rabbits; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000502:physiology; D012867:Skin / Q000166:cytology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*